Abstract In the nearly 20 years since the groundbreaking sequencing of the human genome, the potential for precision genetic medicine has still not been realized. More recently, novel technologies such as CRISPR-Cas gene editing, which relies on DNA-cleaving nucleases discovered in bacteria, enable precise enzymatic editing of the human genome. Although this technique opens up opportunities to prevent or cure many diseases with unmet medical need, before this powerful technology can be translated into medical therapies it must be shown to be not only effective but safe. Off-target editing events with CRISPR-Cas systems occur due to prolonged nuclease activity resulting in unwanted side effects of the gene editing process, including the potential for oncogenic mutations or cellular toxicity. These safety issues can be addressed by designing and implementing more effective ways to control the gene editing process. Acrigen Biosciences is developing a solution to bring safe in vivo CRISPR-Cas gene editing to the market for the first time. This innovative approach encompasses robust anti-CRISPR small-protein off-switches for DNA-cleaving nucleases. This robust and universal approach to temporal control of the gene editing process will be key in translating the potential of CRISPR-Cas to the clinic, and aligns well with NCATS? mission. The ultimate goal of this work is to identify proteins that are efficacious inhibitors of a Cas9 ortholog in human cells. The market for safe gene editing approaches will be very large: the technology can be applied to various monogenic diseases, cancers, and infectious diseases. The Phase I project has 3 specific aims. (1) Establish cell-based and in vitro quantitative experiments to assess inhibition in the function and potency of a Cas9 ortholog by (a) developing a candidate inhibitor screening platform and (b) using biochemical assays to characterize the mechanisms of anti-CRISPR proteins. Milestone: Titratable nuclease expression, as measured by Western blot and bacteriophage targeting in bacteria, looking at phage replication titers as a proxy for CRISPR activity. We will quantify the activity of candidate inhibitors with in vitro DNA-binding and DNA-cleavage experiments. (2) Identify candidate anti-CRISPRs by (a) detecting self-targeting in bacteria encoding homologs of a Cas9 ortholog and (b) identifying bacteriophages that are recalcitrant to the action of a Cas9 ortholog. Milestone: Select ~100 candidate proteins which are small in size, encoded by bacterial mobile elements, and associated with known anti-CRISPR marker genes. The discovery will be based on bioinformatics and phage-based approaches, such as phage replication quantification when targeted by CRISPR. Candidates to be tested in quantitative experimental system established in Aim 1. (3) Assess anti-CRISPR efficacy in two human cell lines (HEK293T and U2-OS-EGFP). Milestone: Identify 5 non-homologous proteins that can inhibit 90% of gene editing in model human cells, using a Cas9 ortholog to edit genes at three different loci.